Human African Trypanosomiasis is returning to levels not seen since the early 20th century. Animal trypanosomiasis remains a significant problem and is a major source of human infection with the virulent Trypanosoma brucei rhodesiense. New drugs are urgently required for all forms of Trypanosomiasis and Leishmaniasis, and understanding the genetic and metabolic circuits of trypanosomes is an important component of the process of validating targets for drug development. Trypanosomes have also attracted general scientific interest as the most widely studied 'differently evolved' eukaryote, with a proven record of contributing important discoveries in membrane biology, cell signaling, telomere biology and gene expression. T. brucei has the potential to continue to contribute to understanding aspects of eukaryotic gene regulation, chromatin modification and telomere structure that are of great contemporary interest. First-generation tools for the genetic manipulation of trypanosomes, pioneered by several laboratories including my own, have severe limitations-of which investigators in the field are increasingly aware-for large-scale analysis of the organism in the context of the soon-to-be-completed genome project and the opportunities that it presents for major advances in understanding the biology of these organisms. A substantial effort is required to develop tools to effectively utilize the genome sequence and to regain momentum in the study of trypanosomes. The aim of this proposal is to improve upon existing cell lines, vectors and protocols, and to develop new approaches, including insertional mutagenesis, for trypanosome genetics. Tools for forward genetics, allowing genome-wide screens by either transposon mutagenesis or the use of RNAi libraries, will become increasingly important to identify the novel genes that undoubtedly regulate the circuitry that is critical to the unique life style, pathogenicity and virulence, of trypanosomes, and which cannot be identified by bioinformatics approaches.